Pardanani, Animesh

Abstract Abstract 4104 Background: The International Prognostic Scoring System (IPSS) for primary myelofibrosis (PMF) uses five predictors of inferior survival: age > 65 years, hemoglobin < 10 g/dL, leukocytes > 25 × 109/L, circulating blasts ≥ 1% and constitutional symptoms (Cervantes F et al. Blood 2009; 113: 2895). The dynamic IPSS (DIPSS) utilizes the same prognostic variables but can be applied at any time during the disease course (Passamonti F et al. Blood 2010; 115: 1703). IPSS-independent risk factors for survival have since been described and include unfavorable karyotype, red cell transfusion need and platelets < 100 × 109/L. Objectives: i) To determine if the aforementioned IPSS-independent risk factors for survival in PMF (i.e. unfavorable karyotype, red cell transfusion need and platelets < 100 × 109/L) are also DIPSS-independent. ii) To develop and validate a refined DIPSS model that incorporates DIPSS-independent prognostic factors for survival. iii) To establish a prognostic model for leukemia-free survival in PMF. Methods: The Mayo Clinic database for PMF was used to identify patients in whom bone marrow histologic and cytogenetic information was obtained at the time of their referral. WHO criteria were used for PMF diagnosis and leukemic transformation (Vardiman JW et al. Blood 2009; 114: 937). None of the patients in the current study were included in the original group of patients used to describe DIPSS (Passamonti F et al. Blood 2010; 115: 1703). Results: A total of 793 patients met the above-stipulated criteria. The study population was divided into two groups based on whether or not patients were seen at the Mayo Clinic within (n=428; training set) or beyond (n=365; test set) their first year of diagnosis. i) Objective 1: Multivariable analysis that included DIPSS risk category (low, intermediate-1, intermediate-2 and high risk), karyotype (favorable or unfavorable), platelet count (≥ or <100 × 109/L) and transfusion status (independent or dependent) was performed using the training set and all four variables were found to be significant as independent predictors of survival (p<0.0001, <0.0001, 0.0008 and 0.01, respectively). ii) Objective 2: Using the respective hazard ratios (HR) obtained during the multivariable prognostic analysis in the training set, HR-weighted adverse points were assigned: 3 points for high-risk DIPSS, 2 points for intermediate-2 risk DIPSS, and 1 point each for intermediate-1 risk DIPSS, unfavorable karyotype, platelets < 100 × 109/L or transfusion need. Accordingly, a refined DIPSS model (i.e. DIPSS-plus) was devised using the training set (Figure 1) and subsequently validated in the test set (Figure 2): low-risk (0 adverse points), intermediate-1 risk (1 adverse point), intermediate-2 risk (2-3 adverse points) and high-risk (4-6 adverse points). Considering the entire group of 793 study patients, the respective median survivals were 185, 78, 35 and 16 months (p<0.0001). ii) Objective 3: Multivariable analysis identified platelet count (p=0.0007) and karyotype (p=0.04) but not DIPSS or transfusion status as independent predictors of leukemia-free survival. We used these two variables to classify patients into a low (favorable karyotype and platelets ≥ 100 × 109/L) or high (unfavorable karyotype or platelets < 100 × 109/L) risk category for leukemic transformation; the respective 5- and 10-year risk of leukemic transformation were 6% and 12% vs. 18% and 31%, respectively (p<0.0001; HR 3.3, 95% CI 1.9–5.6). Conclusions: DIPSS-plus effectively incorporates prognostic information from karyotype, platelet count and transfusion status into the DIPSS prognostic model for PMF. Unfavorable karyotype or platelets <100 × 109/L is associated with a significantly higher risk of leukemic transformation in PMF. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 4112 We report the characterization of BMS-911543, a potent and functionally selective small molecule inhibitor of the Janus kinase family (JAK) member, JAK2. BMS-911543 is a reversible inhibitor of JAK2 with a biochemical IC50 of 0.001 μ M and Ki of 0.48 nM. It has over 74- and 350-fold selectivity against the other JAK family members, JAK3 and JAK1, respectively. Further, examination of > 450 other kinases did not reveal significant inhibitory activity for this JAK2 inhibitor. Functionally, BMS-911543 displayed potent anti-proliferative and pharmacodynamic (PD) effects in mutated JAK2-expressing cell lines dependent upon JAK2-STAT signaling and had little activity in cell types dependent upon other pathways such as JAK1 and JAK3. BMS-911543 was evaluated in colony growth assays using primary progenitor cells isolated from patients with JAK2V617F-positive myeloproliferative disease (MPD) and resulted in an increased anti-proliferative response in MPD cells as compared with those from healthy volunteers. Similar to these in vitro observations, BMS-911543 was also highly active in in vivo models of JAK2-pSTAT signaling in multiple species (mouse, rat, dog and monkey) with sustained pathway suppression being observed after a single oral dose. Additionally, BMS-911543 was evaluated for effects in a JAK2V617F-expressing SET-2 xenograft model system and displayed a minimally effective dose of <2 mg/kg on pSTAT5 pathway suppression, which lasted up to 8 hours. BMS-911543 was also compared to pan-JAK inhibitors in a mouse model of immunosuppression. At low dose levels active in JAK2-dependent PD models, no effects were observed on antigen-induced IgG and IgM production whereas a pan-JAK family inhibitor showed pronounced effects at all dose levels tested. The mechanistic selectivity of BMS-911543 to pan-JAK family inhibitors was extended through comparative analysis of these inhibitors in whole genome gene expression profiling experiments performed in sensitive cell types. In this comparison, BMS-911543 modulated a distinct subset of transcriptional changes as compared to pan-JAK inhibitors, thereby defining a minimal set of transcriptional changes underlying the pharmacologic effects of JAK2 inhibition. Collectively these results define the mechanistic basis for a differential therapeutic index between selective JAK2 and pan-JAK family inhibition pre-clinically and suggest a therapeutic rationale for the further characterization of BMS-911543 in patients with MPD and in other disorders characterized by constitutively active JAK2 signaling. Disclosures: Purandare: Bristol-Myers Squibb: Employment. McDevitt:Bristol-Myers Squibb: Employment. Gottardis:Bristol-Myers Squibb: Employment. You:Bristol-Myers Squibb: Employment. Lombardo:Bristol_Myers Squibb: Employment. Penhallow:Bristol-Myers Squibb: Employment. Vuppugalla:Bristol-Myers Squibb: Employment. Trainor:Bristol-Myers Squibb: Employment. Lorenzi:Bristol-Myers Squibb: Employment.

Abstract Abstract 460 Background: CYT387 is a potent JAK-1/2 inhibitor that suppresses the in vitro growth of cells harboring JAK2V617F (Leukemia 2009;23:1441) and was effective in a murine model of myeloproliferative neoplasms (MPN) (Blood 2010;115:5232). Aims/Methods: To assess the safety, tolerability, and pharmacokinetic behavior of CYT387 in a Phase I dose-escalation study in patients with high- or intermediate-risk primary myelofibrosis (PMF) and post-PV or post-essential thrombocythemia (ET) myelofibrosis. The secondary objective was evaluation of preliminary efficacy. CYT387 was administered orally once daily in 28-day cycles. Once dose-limiting toxicity (DLT) was identified, a dose-confirmation cohort initiated treatment at the maximum tolerated dose (MTD) or lower. Result: Thirty six subjects (median age 64 years) have been enrolled (targeted accrual 120); 18 each in the dose escalation and dose confirmation phases. Twenty-three subjects had PMF, 8 post-PV MF, and 5 post-ET MF; 81% were JAK2V617F-positive. Median palpable spleen size was 18 cm and 20 subjects (56%) were red cell transfusion-dependent at study entry. Prior treatment included JAK inhibitors (9 and 1 subjects with INCB018424 and TG101348, respectively) and pomalidomide in 9 patients. The median treatment duration to date is 15 weeks (range 4–38). Dose-linear plasma exposures were observed up to 300 mg/day, with mean elimination T1/2 at steady state ranging from 3.9 to 5 hours across doses. Toxicity: All 36 subjects were evaluable for toxicity. At 400 mg/day, 2 of 6 subjects experienced DLT (1 each with asymptomatic grade 3 hyperlipasemia and grade 3 headache that were reversible upon holding drug); consequently, the MTD was declared at 300 mg/day. In the dose-confirmation phase, subjects were started at one of 2 dose levels that were deemed clinically effective: 150 mg/day (n=15) and 300 mg/day (n=3). Thirty-five subjects are currently on active therapy: 100 mg/day (n=2), 150 mg/day (n=20), 300 mg/day (n=10), and 400 mg/day (n=3). CYT387 was well tolerated. No grade 4 non-hematological toxicities were observed. Grade 3 non-hematologic adverse events were infrequent and included increased transaminases (n=2), increased alkaline phosphatase (n=2), headache/head pressure (n=2), increased lipase (n=1), and QTc prolongation (n=1). Thirteen (36%) subjects experienced “first-dose effect” characterized by grade 1 lightheadedness and hypotension; this phenomenon was self-limited and generally resolved within 3–4 hours with rare recurrence. Grade 3/4 thrombocytopenia was seen in 8 (22%) subjects, and treatment-emergent grade 3 anemia was seen in 1 subject only (3%). Treatment-emergent grade 3/4 neutropenia was not observed. Efficacy: Thirty two of 36 subjects who completed at least 1 cycle were eligible for response assessment: Anemia: Twenty two subjects were evaluable for anemia response (baseline Hgb <10 g/dL or red cell transfusion-dependent). Of these, 9 subjects (41%) achieved the threshold of response for “Clinical Improvement (CI)” per the International Working Group for MPN Research and Treatment (IWG-MRT) criteria, including 2 of 4 subjects who were previously treated with INCB018424. An additional 5 subjects experienced a >50% reduction in transfusion requirement, thus increasing the total anemia response rate to 63%. Splenomegaly: Thirty of 32 evaluable subjects had splenomegaly at baseline: median 20 cm; range 10–32 cm. Twenty nine subjects (97%) had some degree of spleen size reduction (median 9 cm; range 2–18 cm): 11 (37%) patients have achieved a minimum 50% decrease in palpable spleen size, thus qualifying them for a CI, including 3 of 8 subjects (38%) who were previously treated with INCB018424. Constitutional symptoms: The proportion of patients with the following symptoms at baseline, are: fatigue (97%), pruritus (22%), night sweats (38%), cough (13%), bone pain (28%), and fever (16%). At last follow up, improvement (complete resolution) in these symptoms was reported by 68% (16%), 86% (57%), 83% (75%), 75% (50%), 78% (44%), and 100% (100%), respectively. Conclusion: CYT387 is first-in-class of the JAK inhibitors with a significant response rate in anemia in myelofibrosis patients. The drug also shows substantial activity in reducing spleen size and controlling constitutional symptoms. CYT387 is well tolerated, and treatment responses have been seen both at (300 mg/day) and below (150 mg/day) the MTD. Disclosures: Pardanani: Cytopia Inc.: Research Funding. Off Label Use: Clinical trial data for CYT387 use in Myelofibrosis. Fida:YM Biosciences Australia: Employment, Equity Ownership. Burns:YM Biosciences Australia: Employment, Equity Ownership. Smith: YM Biosciences Australia: Employment, Equity Ownership.

Abstract Abstract 4094 Background: Our group has previously reported the benefits of thalidomide-prednisone based regimens for improvement of anemia, thrombocytopenia, and splenomegaly for patients with MF; both primary myelofibrosis (PMF), and arising from essential thrombocythemia and polycythemia (Post ET/PV MF). We sought to evaluate the overall efficacy of these commonly used regimens by evaluating long-term outcomes and applying the IWG-MRT response criteria (Tefferi et. al. Blood 2006) which arose after completion of these trials. Methods: We retrospectively analyzed the long-term outcomes, and assessed the initial therapeutic responses using IWG-MRT criteria from three parallel completed trials of MF using a thalidomide (THAL-50 mg/day) and prednisone (3 month taper beginning at 40mg, stopped at 3 months) backbone. The trials had identical eligibility criteria including adequate organ function and need for MF therapy (defined as symptomatic: anemia and/or splenomegaly). The trials were 1) THAL-PRED alone, THAL only after 3 months 2) THAL-PRED-etanercept (ETAN - TNF- alpha inhibitor at 25 micrograms subcutaneously twice a week), THAL and ETAN only after 3 months and 3) THAL-PRED – oral cyclophosphamide (CTX 25mg daily orally), THAL alone after 3 months. Results: Patients: A total of 50 pts were enrolled in these 3 trials (Males n=36, 72%) with median age of 68.5 years (Range 43–85), with 79% having PMF. Patients had advanced disease in general with 88% having intermediate 2 or high risk MF by IWG criteria (Cervantes et. al. Blood 2009), 62% patients with red cell transfusion dependence and 50% with an abnormal karyotype. Therapy initial results: 80 % of patients reached the three month juncture on the trials, with 40% reaching 6 months. Initial toxicity was myelosuppression with 3 cases of grade 3 anemia, 3 cases of grade 3 neutropenia and 4 cases of grade 3 thrombocytopenia. There were no grade 4 or higher hematological toxicities noted. Initial neuropathy was uncommon and seen in only 4% of patients. No complete responses were observed and only 1 patient had partial response (by IWG-MRT criteria). 14 patients (28% overall) met the new IWG-MRT criteria for clinical improvement; 11 for anemia (22%), 2 for thrombocytopenia (4%) and 3 for splenomegaly (6%). Responses occurred relatively quickly at an overall median of 8 weeks (range 4–12) after enrollment. Long Term Outcomes: After a median follow-up of 36 months across this cohort we observed an overall median duration of response of 8.5 months (range 3–42). Responses to THAL based regimens can lead to periods of prolonged stabilization after cessation of therapy. We observed an overall median time to institution of next therapy of 3 months (range 1–50). At the time of this analysis 14 patients (28%) have expired of their MF and median survival across the entire cohort was 36 months (3-106). Comparison of Regimens: Comparison of these three independent trials (Table 1) suggests greater toxicity and inferior response rate, duration of response, and time to next therapy with the cyclophosphamide containing regimen. Conclusion: THAL-PRED based regimens are active in a subset of MF patients for therapy primarily of anemia, and for some patients a response of good duration (even after cessation of therapy) may be obtained. IWG-MRT response assessment demonstrates utilizing additional agents to a THAL-PRED regimen do not appear to augment (and may detract from responses). Newer agents such as pomalidomide may have similar to greater efficacy without neuropathy and less myelosuppression. Disclosures: Off Label Use: There is no FDA approved agent in Myelofibrosis. All the drugs discussed; Thalidomide, Prednisone, Etanarcept and Cyclophosphamide are off-label. Mesa:SBio: Research Funding; Novartis: Research Funding; Celgene: Research Funding; Incyte: Research Funding; Roche: Research Funding; eisai: Research Funding; telik: Research Funding.

Abstract Abstract 4122 Background: We have previously identified sole +9, 13q- or 20q- as “favorable” and sole +8 or complex karyotype as “unfavorable” cytogenetic abnormalities in primary myelofibrosis (PMF) (Blood 2010; 115: 496). The purpose of the current study, which includes more than twice the number of patients included in previous studies, was to identify additional prognostically-relevant cytogenetic abnormalities in PMF and refine cytogenetic risk categorization for overall and leukemia-free survival. Methods: Clinical and laboratory data were collected from consecutive patients with PMF seen at our institution and in whom cytogenetic information at or within 1 year of diagnosis was available. Diagnosis of PMF and acute myeloid leukemia were according to the World Health organization (WHO) criteria. Results: A total of 433 patients with PMF were included in the current study. Median age at diagnosis was 65 years. IPSS risk distributions were low in 12% of patients, intermediate-1 in 25%, intermediate-2 in 24% and high in 39%. JAK2V617F mutational frequency was 60%. Cytogenetic findings were normal in 275 (64%) patients. Among the 158 (36%) patients with abnormal karyotype, 109 (69% of abnormal cases) represented sole abnormalities, 23 (15%) two abnormalities and 26 (17%) three or more (i.e. complex) abnormalities. In an effort to identify cytogenetic categories of similar prognosis, each one of 12 operational cytogenetic categories was separately compared with both normal and complex karyotype. Accordingly, we were able to devise a two-tired cytogenetic risk stratification with highly significant differences in overall and leukemia-free survival (Figures 1 and 2): unfavorable (complex karyotype or sole or two abnormalities that include +8, -7/7q-, i(17q), inv(3), -5/5q-, 12p- or 11q23 rearrangement) and favorable (all other cytogenetic findings including normal karyotype). Median survivals of patients with favorable and unfavorable karyotype were 5.2 and 2.0 years, respectively (p<0.0001). The corresponding 5-year survival rates were 51% and 8% (HR 3.1, 95% CI 2.2–4.3; p<0.0001). Multivariable analysis confirmed the IPSS-independent prognostic value of cytogenetic risk categorization (p<0.0001; HR 2.1, 95% CI 1.5–3.1) and also identified thrombocytopenia (platelet count < 100 × 109/L) as another independent predictor of inferior survival (p<0.0001; HR 1.9; 95% CI 1.4–2.6). A similar multivariable analysis showed that cytogenetic risk profile (p=0.001; HR 4.1, 95% CI 1.7–9.6) and platelet count (p=0.04; HR 2.3, 95% CI 1.0–5.0), but not IPSS (p=0.27), predicted leukemia-free survival; the 5-year leukemic transformation rates for unfavorable and favorable karyotype were 46% and 7%, respectively (HR 5.5, 95% CI 2.5–12.0; p<0.0001). These results, in terms of both overall and leukemia-free survival analysis, did not change when patients receiving allo-SCT were censored at the time of their transplant. Among patients with favorable karyotype; the incidences of leukopenia and thrombocytopenia were highest in patients with sole 20q- (41% and 38%, respectively) and lowest in those with sole 13q- (0% and 0%, respectively). Conclusions: The current study provides the rationale and necessary details for incorporating cytogenetic findings and platelet count in future prognostic models for PMF. The study also revealed a highly significant association between sole 20q- and both leukopenia and thrombocytopenia, which suggests a 20q- haploinsufficient gene effect. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 4105 Background: JAK2 mutations are found in approximately 99% of patients with polycythemia vera (PV) and 60% of those with essential thrombocythemia (ET) or primary myelofibrosis (PMF). It is currently assumed that other mutations relevant to JAK signaling contribute to the pathogenesis of JAK2 mutation-negative myeloproliferative neoplasms (MPN). The same might hold true for some cases of “idiopathic erythrocytosis” associated with subnormal serum erythropoietin level (sEpo). LNK is a plasma membrane-bound adaptor protein whose function includes inhibition of wild-type and mutant JAK2 phosphorylation. LNK exon 2 mutations were recently described in two patients with JAK2V617F-negative ET or PMF. Both mutations involved the LNK pleckstrin homology (PH) domain; one was a 5 base-pair deletion and missense mutation leading to a premature stop codon (603_607delGCGCT; 613C>G) and the other a missense mutation (622G>C; glutamic acid to glutamine substitution; E208Q). Objectives: i) To estimate the prevalence of LNK mutations in chronic and blast phase MPN ii) To determine if LNK mutations are mutually exclusive of other MPN-associated mutations iii) To test the hypothesis that LNK mutations might contribute to the pathogenesis of JAK2 mutation-negative PV or otherwise unexplained erythrocytosis Methods: LNK mutation analysis was performed on bone marrow or blood cells using modified primers for amplifying the SH2 and PH domains, using previously published methods (Oh et al. Blood First Edition Paper, prepublished online April 19, 2010; DOI 10.1182/blood-2010-02-270108). Results: LNK mutation studies were performed in 172 patients; paired chronic-blast phase samples were analyzed in 26 cases. Diagnoses in the 172 study patients were as follows: 78 chronic-phase MPN (39 JAK2V617F-positive PV, 25 PMF and 14 ET) enriched for TET2, IDH, JAK2V617F, or MPL mutations, 61 blast-phase MPN (41 blast-phase PMF, 11 blast-phase PV and 9 blast-phase ET), 25 JAK2V617F-negative PV and 8 “unexplained erythrocytosis” associated with subnormal sEpo and negative for Epo receptor (EPOR) mutations. Ten novel heterozygous LNK mutations, all but one affecting exon 2 in the PH domain, were identified: 6 missense mutations involving codons 215, 220, 223, 229 and 234, one nonsense mutation involving codon 208 (622G>T leading to glutamic acid to a stop codon substitution; E208X), one synonymous mutation involving codon 208, and 2 deletion mutations involving exon 2 (685-691_delGGCCCCG) or exon 5 (955_delA). LNK mutations were most frequent in blast-phase MPN, occurring in 6 (9.8%) of the 61 informative patients; chronic-phase sample analysis in 4 of these revealed the same mutation in only one case. Mutant LNK was detected in chronic-phase samples only in 2 additional patients with blast-phase MPN. Among the 8 LNK-mutated blast-phase MPN cases, 7 had blast-phase PMF. The clinical phenotype in the remaining 2 LNK-mutated patients was that of an isolated erythrocytosis without overt features of PV. JAK2V617F was documented in 3 and IDHR140Q in 1 LNK-mutated patients. Conclusions: LNK mutations i) target an exon 2 ‘hot spot' in the PH domain spanning residues E208-D234, ii) might be most prevalent in blast-phase PMF iii) are not mutually exclusive of JAK2 or IDH mutations and iv) might be part of the “missing link” in the pathogenesis of JAK2 mutation-negative “idiopathic” erythrocytosis. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 459 Background: A Phase I study of TG101348, a potent, oral JAK2-selective inhibitor in patients with high- or intermediate-risk primary, post-polycythemia vera (PV) and post-essential thrombocythemia (ET) myelofibrosis (MF) recently completed enrollment. Data from the dose escalation cohort (n=28; 30–800 mg administered as a single daily dose) showed dose-linear plasma exposure, with mean elimination T1/2 at steady state ranging from 16 to 34 hours. The dose-limiting toxicity was reversible asymptomatic grade 3 or 4 amylasemia/lipasemia; the maximum tolerated dose was 680 mg. We now present updated results from an extension study in which subjects continued treatment beyond six 28-day cycles. Result: In the entire study, 59 subjects (median age 64 years) were treated. Forty four patients had PMF, 12 post-PV MF, and 3 post-ET MF; 86% were JAK2V617F-positive. Median palpable spleen size was 18 cm and 21 (36%) patients were transfusion-dependent at study enrollment. Forty three (73%) subjects continued treatment on the extension study; the median (range) cumulative exposure to TG101348 was 380 days (170-767); the number of cycles of treatment completed ranged from 7–29, and the proportion of subjects who completed 12, 18, and 24 cycles of treatment was 72%, 28%, and 14%, respectively. The median (range) treatment dose at data cutoff was 440 mg/day (120 to 680 mg/day; n = 35). Toxicity: TG101348 was well tolerated. Non-hematologic, at least possibly related adverse events included nausea (69%; Grade 3=3%), diarrhea (64%; Grade 3=10%), and vomiting (58%; Grade 3=3%), that were dose-dependent; the symptoms were self-limited or controlled with symptomatic treatment and/or dose reduction. Also observed were asymptomatic increases in serum lipase (27%), AST/ALT (27%/25%), creatinine (24%), and alkaline phosphatase (17%). Grade 3/4 hematological adverse events possibly related to TG101348 included treatment-emergent anemia (35% of patients who were not transfusion dependent at baseline), thrombocytopenia (24%), and neutropenia (10%). Spleen: During cycles 1–6, 42% of patients achieved Clinical Improvement (CI) per the International Working Group for Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) criteria for spleen response. The CI rate for the 43 subjects who continued treatment beyond 6 cycles was 65%, and the median time (range) to CI was 140 days (77-802). After 12 cycles, 36 (95%) of the 38 evaluable patients had some degree of spleen size reduction; 32 (85%), 26 (68%), and 10 (26%) of subjects had a minimum 25%, 50%, and 100% decrease in palpable splenomegaly. JAK2V617F allele burden: Of the 48 JAK2V617F mutation-positive subjects, 23 (48%) had a baseline mutant allele burden of >20%. Of these, 20 subjects with a baseline median allele burden (range) of 62% (23% to 100%) completed at least 6 cycles of treatment. Sixteen subjects (80%) exhibited a median 62% (range 6% to 96%) decrease in JAK2V617F burden, including 9 (45%) with a ≥50% decrease; 4 subjects (20%) had increases (range 18% to 58%). Eighteen subjects (78%) completed 12 cycles of treatment; 13 (72%) had a median 50% decrease (range 29% to 82%), including 7 (39%) subjects with a ≥50% decrease; 5 subjects (28%) had no change or increases (range 0% to 22%) in JAK2V617F. Constitutional symptoms: For patients with data at baseline and cycle 6, early satiety, fatigue, night sweats, cough, and pruritus were reported by 56%, 47%, 24%, 22%, and 19% of subjects, respectively. Improvement (complete resolution) rates observed at cycle 6 were: 75% (40%), 65% (29%), 89% (89%), 88% (75%), and 100% (57%) for the aforementioned symptoms, respectively. Leukocytosis/Thrombocytosis: Leukocytosis was present at baseline in 33 subjects; following 6 and 12 cycles of therapy, 57% (n=28) and 56% (n=25) achieved normal WBC counts. Thrombocytosis was noted at baseline for 10 (17%) subjects; following 6 and 12 cycles of therapy, 90% (n=10) and 88% (n=8) achieved normal platelet counts. Conclusion: The current study provides proof-of-concept that selective JAK2 inhibition has anti-myeloproliferative/anti-clonal activity in myelofibrosis. The majority of subjects treated with TG101348 for more than 6 cycles experienced a rapid, significant, and durable decrease in splenomegaly, normalization of leukocyte/platelet counts, and improvement of disease-related symptoms, all within the context significantly decreased JAK2V617F burden. Disclosures: Off Label Use: There is no FDA approved drug for Myelofibrosis.All the drugs discussed will be off-label. Stone:Novartis: Consultancy. Silverman:TargeGen: Consultancy. Shorr:TargeGen: Employment, Equity Ownership.

Abstract Background: SL-401 is a novel targeted therapy directed to the interleukin-3 receptor (CD123), a target overexpressed by many hematologic malignancies. SL-401 is currently being advanced through clinical trials in patients with a variety of CD123+ malignancies including blastic plasmacytoid dendritic cell neoplasm (BPDCN), acute myeloid leukemia (AML) in remission with high relapse risk, and multiple myeloma. CD123 has been shown to be expressed on myeloproliferative neoplasm (MPN) cells as well as microenvironmental immune cells, namely plasmacytoid dendritic cells (pDCs), in the bone marrows of some patients with MPN including chronic myelomonocytic leukemia (CMML). Microenvironmental pDCs have been implicated in promoting plasma cell disorders and preliminary data suggest that pDCs could play a related role in some myeloid neoplasms. Accordingly, a therapy directed at both CD123-expressing myeloid cells and neighboring CD123-expressing pDCs could provide therapeutic benefit. SL-401 is being evaluated in patients with advanced, high-risk MPN, including systemic mastocytosis (SM), myelofibrosis (MF), primary eosinophilic disorders (PED), and CMML. Preliminary results are reported here. Methods & Results: This multicenter, single-arm Phase 2 trial of patients with advanced, high risk MPN includes a lead-in (stage 1) and expansion (stage 2). In stage 1, patients receive SL-401 as a daily IV infusion at 7, 9, 12 ug/kg/day, or higher for days 1-3 of a 21-28 day cycle in a 3x3 design. In stage 2, patients receive SL-401 at the dose determined in stage 1. Study objectives include characterization of the safety profile, including determination of the maximum tolerated or tested dose, and detection of preliminary efficacy signals including evaluation of tumor response and progression free survival by standard criteria. As of 7/20/16, 6 patients with MPN (MF, n=3; CMML-2, n=2; CMML-1, n=1) received SL-401 (7 ug/kg, n=3; 9 ug/kg, n=3). The median age was 66 years (range: 42-81 years). Patients treated at all doses received 1+ to 2+ cycles (ongoing) of SL-401. The most common treatment-related adverse events (AEs) were thrombocytopenia (2/6; 33%) and fatigue (2/6; 33%); the most common ≥ Grade 3 treatment-related AEs were thrombocytopenia (2/6; 33%) and anemia (1/6; 17%); there has been no DLT. Efficacy assessments are ongoing. Patients are currently enrolling in the 12 ug/kg/day cohort. Conclusions: Initial dosing of SL-401 appears to be well-tolerated in patients with MPN and CMML, with no unexpected AEs observed. Given CD123 expression on myeloid neoplastic cells as well as microenvironmental immune cells (namely, pDCs), SL-401 may offer a novel, targeted therapeutic approach in patients with high-risk MPN and CMML of unmet medical need. Enrollment continues in stage 1 of this ongoing Phase 2 trial and updated safety and efficacy data will be presented. Clinical trial information: NCT02268253. Disclosures Gupta: Novartis: Consultancy, Honoraria, Research Funding; Incyte Corporation: Consultancy, Research Funding. Carraway:Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Baxalta: Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees. Schiller:Incyte Corporation: Research Funding. Talpaz:Incyte Corporation: Other: Travel expense reimbursement, Research Funding; Ariad: Other: Expense reimbursement, travel accomodation expenses, Research Funding; Novartis: Research Funding; Pfizer: Consultancy, Other: travel accomodation expenses, Research Funding. McCloskey:Novartis: Speakers Bureau; Incyte: Consultancy; Ariad: Consultancy, Speakers Bureau; Amgen: Speakers Bureau. Lee:Alexion Pharmaceuticals: Consultancy; Amgen: Consultancy; Baxalta: Consultancy; Boehringer Ingelheim: Consultancy; Pfizer Inc: Consultancy. Yacoub:Alexion: Honoraria; Seattle Genetics: Consultancy, Honoraria, Speakers Bureau; Incyte: Consultancy, Honoraria, Speakers Bureau. Ali:Incyte Corporation: Research Funding. Chen:Stemline Therapeutics, Inc.: Employment, Equity Ownership. Shemesh:Stemline Therapeutics: Employment, Equity Ownership. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties.

Abstract Abstract 627 Myeloproliferative diseases (MPD) are clonal hematologic disorders that present with increased numbers of functional, mature, terminally differentiated myeloid elements. Even though genetic, biochemical and functional studies have provided important insights into the pathogenesis of MPDs, the role of epigenetic changes in disease pathobiology is not well elucidated. We performed the HELP assay to study genome-wide methylation patterns in cases of Polycythemia Vera (PV), Essential Thrombocytosis (ET) and Idiopathic Myelofibrosis (IMF) and compared it with normal matched controls. The HELP assay uses differential methylation specific digestion by HpaII and MspI followed by amplification, two color labeling and hybridization to quantitatively determine individual promoter CpG methylation of 25636 loci. Analysis of 26 MPD neutrophil samples comprising 9 cases of ET, 6 cases of PV and 11 Cases of IMF was performed and compared to normal healthy controls. Unsupervised clustering based on global methylation profiles showed that IMF cases formed a distinct epigenetic cluster, while PV and ET cases were more similar to the normal controls. Further analysis of epigenetic differences between these groups showed that PV and ET samples were characterized by aberrant hypermethylation when compared to controls and had 143 genes that were uniformly hypermethylated. These genes are involved in pathways regulated by the NF-Kb and HNF-4alpha transcription factors. IMF on the other hand was characterized by both aberrantly hyper (n=162) and hypomethylated (n=95) loci when compared to controls. The pathways affected by hypomethylated genes were involved in cytokine cell signaling and MAP kinases. We subsequently validated these observations in an independent set of 8 IMF cases compared to 8 age matched controls. Methylation profiles obtained from whole blood by the HELP assay were able to clearly separate IMF cases from controls demonstrating the validity of our observations. These changes were quantified on a whole genome level by the Luminometric methylation assay (LUMA) that also revealed significantly more hypomethylation in IMF when compared to PV and ET cases (49% hypomethylation in IMF vs. 38% in PV/ET, p<0.05). Selected changes were validated by Mass Array sequencing. We next wanted to determine the epigenomic effects of Jak2V617F mutation and compared methylation profiles of MPD cases with and without the mutation. We observed that cases with Jak2 mutation had a higher number of differentially hypomethylated loci. This striking difference was seen by the LUMA assay also with 67% hypomethylation seen in mutant cases when compared to 48% in those without the mutation (p=0.02). This observation was validated in vitro in a cell line (FDCP) that expressed wither WT or mutant Jak2 kinase. We observed that expression of the mutant kinase led to global hypomethylation. This observation builds on recent data demonstrating nuclear binding of the mutant Jak2 kinase and its effects on the histone epigenetic machinery. In conclusion, we report that MPDs are characterized by various novel epigenetic alterations that affect important functional pathways and IMF is grossly epigenetically distinct from ET and PV. The Jak2 mutation also affects the methylome and leads to global hypomethylation that potentially contributes to the genomic instability and disease pathobiology. Disclosures: No relevant conflicts of interest to declare.

Abstract Background : Anemia is one of the most prominent symptoms in primary myelofibrosis (PMF) and is often associated with inferior quality of life and survival. Current drugs, including JAK inhibitors, are suboptimal in the treatment of PMF-associated anemia and better information on its pathogenesis is critical for the development of more effective drugs. In the current study of JAK2/CALR/MPL annotated patients with PMF, we examined its correlation with both "driver" and "non-driver mutations", as well as cytogenetic abnormalities, in order to gain better insight into its pathogenesis. Methods: Study patients were selected based on availability of "driver" mutation information. PMF diagnosis was according to World Health Organization criteria (Blood. 2009;114:937). Previously published methods were used for CALR, JAK2 and MPL mutation analyses and determination of CALR variants (Blood. 2014;124:2465). Considering their relatively high mutational frequency in PMF, subsets of patients were also screened for ASXL1, spliceosome component (SF3B1, U2AF1, SRSF2, ZRSR2) and TET2 mutations. Cytogenetic analysis and reporting was done according to the International System for Human Cytogenetic Nomenclature. Statistical analyses considered clinical and laboratory parameters obtained at time of first referral at the Mayo Clinic. Results : Analysis was conducted on 722 patients (median age 64 years; 64% males). DIPSS-plus risk distribution was 14% low, 17% intermediate-1, 37% intermediate-2 and 33% high. All patients were annotated for JAK2/CALR/MPL mutations as well as CALR variants; 477 harbored JAK2, 139 CALR and 41 MPL mutations; 65 patients were triple-negative. The 139 CALR -mutated patients included type 1/type 1-like (n =115) and type 2/type 2-like (n =24). Non-driver mutations screened included ASXL1 (n =480; mutated 38%), SRSF2 (n =474; mutated 14%), U2AF1 (n =457; mutated 16%), SF3B1 (n =328; mutated 8%), ZRSR2 (n =180; mutated 11%) and TET2 (n =180; mutated 18%). Karyotype was normal in 60%, favorable in 28% and unfavorable in 12%. Anemia was defined as being absent (normal sex-adjusted hemoglobin level; n =110; 15%), mild (hemoglobin level of ≥10 g/dL but below sex-adjusted normal value; n =263; 36%), moderate (hemoglobin level below 10 g/dL but not transfusion-dependent; n =108; 15%) and severe (transfusion-dependent anemia; n =241; 33%). Presence of at least mild anemia was associated with abnormal karyotype (p=0.006) with no difference between favorable and unfavorable abnormalities, U2AF1 (p=0.002), TET2 (p=0.02) and ASXL1 (p=0.04) mutations; other significant associations included male sex and older age. Presence of moderate to severe anemia was associated with U2AF1 (p<0.0001), SRSF2 (p=0.007) and driver mutations other than CALR type 1/type 1-like (p<0.0001). Presence of severe anemia was associated with U2AF1 (p<0.0001), SRSF2 (p=0.003) and non-CALR driver mutations (17% incidence in both types of CALR variants vs 51% in triple negative, 35% JAK2, 39% MPL mutated cases; p<0.0001). An association with older age but not gender was also noted for both moderate to severe and severe anemia (p<0.0001). During multivariable analysis, independent associations with moderate to severe anemia were confirmed for U2AF1 (p<0.0001), SRSF2 (p=0.007) and age (p=0.0001), but not driver mutation profile (p=0.30). A similar analysis for severe anemia also identified U2AF1, SRSF2 and age as being significantly relevant. Conclusions : The current study identifies older age and the spliceosomal mutations U2AF1 and SRSF2 as having strong and independent association with moderate to severe anemia in PMF. Targeting the spliceosome machinery or its mutant components offers a potential approach in the treatment of PMF-associated anemia. Disclosures Pardanani: Stemline: Research Funding.